The helical repeat of DNA at high temperature.

The increasing number of studies on thermophilic organisms addressed the question of DNA double helix parameters at high temperature. The present study shows that the helix rotation angle per base pair omega of an unconstrained DNA decreases linearly upon temperature increase, up to the premelting range. In the ionic conditions tested, this rule extends to temperatures up to 85 degrees C, which is a common growth temperature for many hyperthermophilic organisms. In addition, the torsional constant K of DNA decreases with temperature, indicating that the energy required to modify the DNA twist is lower at high temperature. These findings have several implications for people working on the structure and enzymology of DNA at high temperature.     

Calculated pH-dependent population and protonation of carbon-monoxy-myoglobin conformers

X-ray structures of carbonmonoxymyoglobin (MbCO) are available for different pH values. We used conventional electrostatic continuum methods to calculate the titration behavior of MbCO in the pH range from 3 to 7. For our calculations, we considered five different x-ray structures determined at pH values of 4, 5, and 6. We developed a Monte Carlo method to sample protonation states and conformations at the same time so that we could calculate the population of the considered MbCO structures at different pH values and the titration behavior of MbCO for an ensemble of conformers. To increase the sampling efficiency, we introduced parallel tempering in our Monte Carlo method. The calculated population probabilities show, as expected, that the x-ray structures determined at pH 4 are most populated at low pH, whereas the x-ray structure determined at pH 6 is most populated at high pH, and the population of the x-ray structures determined at pH 5 possesses a maximum at intermediate pH. The calculated titration behavior is in better agreement with experimental results compared to calculations using only a single conformation. The most striking feature of pH-dependent conformational changes in MbCO-the rotation of His-64 out of the CO binding pocket-is reproduced by our calculations and is correlated with a protonation of His-64, as proposed earlier.     

Genomic adaptation of prokaryotic organisms at high temperature

One of the central issues of evolutionary genomics is to find out the adaptive strategies of microorganisms to stabilize nucleic acid molecules under high temperature. Thermal adaptation hypothesis gives a link between G+C content and growth temperature if there is a considerable variation of guanine and cytosine content between species. However, there has been a long-standing debate regarding the correlations between genomic GC content and optimal growth temperature (Topt). We urged that adaptation to growth at high temperature requires a coordinated set of evolutionary changes affecting: (i) nucleic acid thermostability and (ii) stability of codon-anticodon interactions. Moreover, in Bacillaceae family we have demonstrated that a higher genomic GC level do not have any role in stabilizing mRNA secondary structure at high growth temperature. Comparative analysis between homologous sequences of thermophilic Thermus thermophilus and mesophilic Deinococcus radiodurans suggests that increased levels of GC contents in the coding sequence corresponding to strand structure of Thermus thermophilus genes have stabilizing effect on the mRNA secondary structure, whereas increased levels of GC contents in coding sequences corresponding to aperiodic structure have destabilizing effect on the mRNA secondary structure. In this perspective, a critical review of thermal adaptation hypothesis is further advocated.     

Inhibition of expression of tomato-ripening genes at high temperature

Abstract. Ripening tomato fruits incubated at 35°C fail to achieve normal pigmentation, soften little and show a marked decline in ethylene evolution. Labelling studies in vivo indicate that protein synthesis continues throughout incubation at 35°C although the spectrum of labelled proteins is different to that observed at 25°C. Translation of mRNAs in vitro shows traces of several 'heat-shock' mRNAs at 35°C and the loss of several others normally found in fruit ripened at 25°C. Using ripening-related cDNA clones as hybridization probes the expression of 12 ripening-related genes was followed during incubation at 25°C and 35°C. In general, there was a marked decline in the amounts of these mRNAs following incubation of ripening fruit at 35°C. In particular, mRNA homologous to pTOM 6, a cDNA clone coding for polygalacturonase, a major cell wall degrading enzyme, showed a rapid decline following incubation at 35°C and after 72-h at elevated temperature was undetectable. There was no recovery of expression during 120 h at 35°C and the application of exogenous ethylene did not overcome the inhibition of ripening or lead to the renewed accumulation of polygalacturonase mRNA. It is proposed that the failure to soften normally at elevated temperature is due, in part, to the suppression of polygalacturonase mRNA and that the inhibition of other facets of ripening at 35°C is due to the inhibition or reduced expression of other, as yet unidentified, ripening-related genes.     

Defect in expression of heat-shock proteins at high temperature in xthA mutants.

Escherichia coli mutants lacking exonuclease III (xthA) are defective in the induction of heat-shock proteins upon severe heat-shock treatment (upshift from 30 to 50 degrees C) but not mild heat-shock treatment (upshift from 30 to 42 degrees C). We show that this defect is due to the xthA mutation by complementation. Furthermore, increasing the gene dosage of xthA+ prolongs the synthesis of heat shock proteins seen after a shift to 42 degrees C. Increasing the gene dosage of htpR+ partially suppresses the defect of xthA mutants in the synthesis of heat-shock proteins at 50 degrees C. When an xthA strain was incubated at 42 degrees C before a shift to 50 degrees C, it was then able to carry out the synthesis of heat-shock proteins at 50 degrees C.     

Effects of air movement on swine at high temperature

Sixty pigs were used to study the effects of air movement (0.05, 0.5, and 1.0 m/s) at constant air temperatures of either 5.6 or 11.1°C above optimum, and at 3 different weight ranges (36–50kg, 54–68 kg, and 73–86 kg) during the growing-finishing stages. An air velocity of 0.5 m/s produced significantly greater weight gain and feed conversion than did 0.05 m/s. An air velocity of 1.0 m/s produced intermediate results.     

DNA cleavage and religation by human topoisomerase II alpha at high temperature.

A common DNA religation assay for topoisomerase II takes advantage of the fact that the enzyme can rejoin cleaved nucleic acids but cannot mediate DNA scission at suboptimal temperatures (either high or low). Although temperature-induced DNA religation assays have provided valuable mechanistic information for several type II enzymes, high-temperature shifts have not been examined for human topoisomerase IIalpha. Therefore, the effects of temperature on the DNA cleavage/religation activity of the enzyme were characterized. Human topoisomerase IIalpha undergoes two distinct transitions at high temperatures. The first transition occurs between 45 and 55 degrees C and is accompanied by a 6-fold increase in the level of DNA cleavage at 60 degrees C. It also leads to a loss of DNA strand passage activity, due primarily to an inability of ATP to convert the enzyme to a protein clamp. The enzyme alterations that accompany the first transition appear to be stable and do not revert at lower temperature. The second transition in human topoisomerase IIalpha occurs between 65 and 70 degrees C and correlates with a precipitous drop in the level of DNA scission. At 75 degrees C, cleavage falls well below amounts seen at 37 degrees C. This loss of DNA scission appears to result from a decrease in the forward rate of DNA cleavage rather than an increase in the religation rate. Finally, similar high-temperature alterations were observed for yeast topoisomerase II and human topoisomerase IIbeta, suggesting that parallel heat-induced transitions may be widespread among type II topoisomerases.     

In vitro translation of archaeal natural mRNAs at high temperature

Abstract Efficient in vitro translation of archaeal natural mRNAs at high (75°C) temperature has been achieved by employing either crude cell lysates or purified ribosomes and soluble proteins of the extreme thermophile Sulfolobus solfataricus . The features of the system are described.     

The effect of temperature variation on biomethanation at high altitude

The aim of the current study was to examine effects of daily temperature variations on the performance of anaerobic digestion. Forced square-wave temperature variations (between 11 and 25, 15 and 28, and 19 and 32 degrees C) were imposed on a bench-scale digester using a mixture of llama-cow-sheep manure in a semi-continuous process. The volumetric biogas production rate, methane yield, and the volatile solid reductions were compared with the results obtained from anaerobic digestion (AD) at constant temperatures. The forced cyclic variations of temperature caused large cyclic variations in the rate of gas production and the methane content. As much as 94-97% of the daily biogas was obtained in the 12h half-cycle at high temperature. The values for volumetric biogas production rate and methane yield increased at higher temperatures. The average volumetric biogas production rate for cyclic operation between 11 and 25 degrees C was 0.22Ld(-1)L(-1) with a yield of 0.07m3CH4kg(-1) VS added (VSadd), whereas for operation between 15 and 29 degrees C the volumetric biogas production rate increased by 25% (to 0.27Ld(-1)L(-1) with a yield of 0.08m3CH4kg(-1) VSadd). In the highest temperature region a further increase of 7% in biogas production was found and the methane yield was 0.089m(3)CH(4)kg(-1) VSadd. The employed digester showed an immediate response when the temperature was elevated, which indicates a well-maintained metabolic capacity of the methanogenic bacteria during the period of low temperature. Overall, periodic temperature variations appear to give less decrease in process performance than a priori anticipated.     

Repression of nitrogen fixation in Klebsiella pneumoniae at high temperature

Clostridium perfringens 11268 CDR (Rifr Tcs), the strain transformed in our experiments, was generated by curing a spontaneous, rifampicin-resistant mutant of C. perfringens 11268 (Rifr Tcr). High-temperature growth yielded tetracycline-sensitive, rifampicin-resistant cells which no longer contained pCW3, a 42.8-kilobase plasmid. The tetracycline-sensitive, rod-shaped cell was then converted to an L-phase variant by growth in the presence of penicillin G (10 micrograms/ml) and 0.4 M sucrose. After several passages, the antibiotic was removed from the medium, and cells continued to grow as L-phase variants. Another large plasmid, pJU124 (38.8 kilobases), which confers tetracycline resistance, was used for transformation. Transformation of L-phase variants of C. perfringens 11268 CDR (Rifr Tcs) was mediated by polyethylene glycol. Transformation frequency is a nonlinear function of DNA concentration. Restriction analysis showed that the plasmid isolated from the transformants was identical to that supplied. Stable L-phase variants do not revert to rod-shaped cells, but autoplasts can be both transformed and reverted.     

The temperature sensitive mutant 72c. II. Accumulation at high temperature of ppGpp and pppGpp in the presence of protein synthesis.

A heat sensitive mutant of E. coli has been analyzed. A shift to restrictive temperature leads to an accumulation of ppGpp and pppGpp in both the parental and the mutant strains (both are relA+). The pool of these compounds is shown to decrease with time after the temperature shift in the case of the parental strain, but remains at the same elevated level in the case of the mutant. The temperature shift of the mutant leads to an apparent reduction of stable RNA synthesis; this inhibition can be released by chloroamphenicol or tetracycline. Gross protein synthesis is more or less unaffected at restrictive temperature. In the parental strain little effect is seen on RNA and protein synthesis after the temperature shift. A relA derivative of the mutant does not show the same inhibition of RNA synthesis at high temperature. Sedimentation analysis suggests that mutant 70S ribosome are more stable, when exposed to a lowered Mg2+ concentration, than are 70S ribosomes from the parental strain. In addition, the relative amounts of the two forms of ribosomal protein S6, which can be obtained on DEAE chromatography (Held et al., 1973), are significantly changed in the mutant.     

A temperature sensitive mutant of Escherichia coli which does not allow replication of RNA phage at a high temperature.

A conditional mutant, referred to as RepR43, was isolated from Escherichia coli W2252 by N-methyl-N'-nitro-N-nitroso-guanidine mutagenesis. Although RepR43 does not permit growth of RNA phage beta at the restrictive temperature, 43 degrees C, cell growth and synthesis of macromolecules such as RNA and protein continue at a somewhat reduced rate. Several lines of evidence indicate that a RepR43 function is indispensable for normal phage RNA replication. In addition, this function appears to be involved in the maintenance of the perpetuated phage genome. The addition of 10% sucrose to the medium at the restrictive temperature resulted in the production of the phage, suggesting that the mutant cell might have an altered membrane organization which interferes with normal viral replication.